It is well established that many normal tissues are maintained and regenerated by a small subset of pluri- (or toti-) potential stem cells (SC) [Weismann, A F L. Das Keimplasma. G. Fischer, Jena, 1892; Dubois, F., C.R. Seances Soc Biol et ses Filiales (Paris) 1948, 142:699-700; Dubois S. F et al., C.R. Seances Soc Biol et ses Filiales (Paris) 1960, 155:115-118; Till J. E et al., Radiat Res 1961, 14: 213-222; Till J. E. et al., Proc Natl Acad Sci USA 1964, 51: 29-36; Lange C. S et al., Int J Radiat Biol. 1968; 14: 373-388; Lange C. S., Exp Geronto 1968; 3: 219-230; Wheldon T. E. et al., Br J Radiol. 1982, 55: 759; Lajtha L. G., Stem Cell Concepts In: Stem Cells, Their Identification and Characterization. Ch. 1, pp 1-11 (Ed. CS Potten) Churchill Livingstone, London, 1983; Lange, C. S., Stem cells in planarians. In: Stem Cells, Their Identification and Characterization. Ch. 3, pp 28-66 (Ed. CS Potten) Churchill Livingstone, London, 1983; Wheldon T. E. et al., Br J Cancer 1986, 53: 382-385; Morrison S. J et al., Immunity 1994, 1: 661-673; Lanzkron S. M. et al., Blood 1999, 93: 1916-1921; Osawa M. et al., Science 1996, 273: 242-245; Bhatia M. et al., Nat Med 1998, 4: 1038-1045; Yilmaz O. H. et al., Blood 2006, 107(3): 924-930; Kiel M. J. et al., Cell 2005, 121: 1109-1121; Kuperwasser C. et al., Proc Natl Acad Sci USA 2004, 101: 4966-4971; Proia D. et al., Nature Protocols 2006, 1: 206-214; Stingl J. et al., Nature 2006, 439: 993-997]. This also holds for human breast development, which can be recapitulated from an organoid and fibroblast mixture in which the former represents 1% or less of the cells [Kuperwasser C. et al., Proc Natl Acad Sci USA 2004, 101: 4966-4971; Proia D. et al., Nature Protocols 2006, 1: 206-214; Stingl J. et al., Nature 2006, 439: 993-997]. However, stem cell identification is still problematic as the marker combinations which purportedly identify prospective hematopoietic stem cells (the best characterized) label a larger fraction (˜2-fold) of cells than that which can repopulate the bone marrow (the functional endpoint) [Yilmaz O. H. et al., Blood 2006, 107(3): 924-930; Kiel M. J. et al., Cell 2005, 121: 1109-1121]; for breast, the fraction is 100-fold [Al-Hajj M. et al., Proc Natl Acad Sci USA 2003, 100: 3983-3988].
The Cancer Stem Cell (CSC) Hypothesis postulates that cancer stem cells 1% or less of the tumor), initially derived from normal tissue stem cells, are necessary for long-term tumor growth and survival and that their eradication is a necessary and sufficient condition for cure [Lajtha L. G., Stem Cell Concepts In: Stem Cells, Their Identification and Characterization. Ch. 1, pp 1-11 (Ed. CS Potten) Churchill Livingstone, London, 1983; Al-Hajj M. et al., Proc Natl Acad Sci USA 2003, 100: 3983-3988; Dick J. E., Proc Natl Acad Sci USA 2003, 100: 3547-3549; Kuperwasser C. et al., Cancer Res 2005, 65: 6130-6138; Jordan C. T., Curr Opinion Cell Biol 2004, 16: 708-712; Bonnet D. et al., Nat Med 1997, 3: 730-737; Singh S. K. et al., Cancer Res 2003, 63: 5821-5828]. This suggestion is not new [Lajtha L. G., Stem Cell Concepts In: Stem Cells, Their Identification and Characterization. Ch. 1, pp 1-11 (Ed. CS Potten) Churchill Livingstone, London, 1983]. Lajtha noted that an organism's cells can be considered to fall into one of three categories (static, transit and stem), the first two of which have no or little proliferative future. The “cell population type which is self-maintaining with extensive proliferation capacity and persists in the body long enough to be compatible with the time scale of carcinogenesis, and therefore satisfies the criteria of being the main potential target for carcinogens, is the stem cell” [Lajtha L. G., Stem Cell Concepts In: Stem Cells, Their Identification and Characterization. Ch. 1, pp 1-11 (Ed. CS Potten) Churchill Livingstone, London, 1983]. Since stem cells can represent less than 0.5% of the total tissue, and DNA synthesis is an error-prone event, it is an excellent strategy for the minimization of cancer risk for most of the divisions to be done by the amplifying transit cells which are soon eliminated. Lajtha further noted that most of the tumor's cells are no longer stem cells, so that the “analytical methods used to study properties of the tumors are measuring properties which may be [those] of the majority of the tumor cell population (which may be responsible for [its] size, but not [its] growth) but not the properties of the stem line which is the minority responsible for the tumor growth.” [Lajtha L. G., Stem Cell Concepts In: Stem Cells, Their Identification and Characterization. Ch. 1, pp 1-11 (Ed. CS Potten) Churchill Livingstone, London, 1983]. In terms of cell kinetics, it is important to distinguish between most of the divisions done by transit cells whose progeny will eventually be eliminated (hence the cell loss factors in tumors [Hall E. J. et al., Radiobiology for the radiologist 6th Edn p 359 Lippincott Williams & Wilkins, Philadelphia, Pa. 2006], and the much smaller number of divisions done by the precursor stem cells which maintain themselves and the supply of transit cells. Hence, kinetically, the stem cells determine treatment outcome. Yet most currently available treatments of cancers ignore this.
Since most cell survival of reproductive integrity assays (the most stringent endpoint) require that the cell be capable of undergoing only 5-6 divisions, as do transit cells in erythropoiesis (6-7 divisions), most such assays do not ask if the bulk of the proliferating (through only a few divisions) tumor cells (even the few tenths of a percent which grow in primary culture) are the tumor stem cells. Consideration of stem cells leads to the conclusion that: assays for tumor sensitivity to potential treatment agents should define clonogenicity as the ability to undergo 10 or more, rather than 5-6 divisions [Puck T. T. et al., J Exp Med 1956, 103: 653-666; Elkind M. M. et al., The Radiobiology of Cultured Mammalian Cells, pp. 69-74, Gordon and Breach, New York, 1967], so as to see the effect on the stem cells rather than on limited-future amplifying-division transit cells [Lajtha L. G., Stem Cell Concepts In: Stem Cells, Their Identification and Characterization. Ch. 1, pp 1-11 (Ed. CS Potten) Churchill Livingstone, London, 1983; Djordjevic B. et al., Acta Oncologica 2006, 45, 412-420].
Potential markers of human cancer stem cells have been reported, e.g., CD44+CD24−/low Lin− as breast cancer markers [Al-Hajj M. et al., Proc Natl Acad Sci USA 2003, 100: 3983-3988]. However, the marker based detection of normal or cancer stem cells in the art can identify cells which are in most cases not stem cells. Much of the recent literature (1990-2008) regarding stem cells and in particular cancer stem cells (CSCs) has been based on antigenic markers used to identify the putative CSCs. The problem with this approach is that these markers do not identify such cells since: 1) the markers do not specify any function of the CSCs, they only help to enrich for subpopulations containing them; 2) cells possessing the appropriate markers (and lacking the inappropriate ones) are only enriched for CSCs and only a fraction of them can initiate a tumor; and 3) some cells lacking the appropriate markers supposedly defining CSCs are also able to initiate a tumor.
Thus, the prior art methods normally enrich, rather than identifying, cancer or normal stem cells. The previous methods, which employ monolayer and agar colony formation did not prevent stromal fibroblast outgrowth from producing artifactual colonies so that the cancer cell (not cancer stem cell) survival curves obtained by those methods were not useful or meaningless for predicting individual patient outcomes. In addition, the earlier methods (e.g., Djordjevic B. et al., Radiat Environ Biophys 1990, 29: 31-46; Lange C. S. et al., Int J Radiat Oncol Biol Phys 1992, 24(3): 511-518; Djordjevic B. et al., Cancer Invest 1991, 9(5): 505-512; Djordjevic B. et al., Cancer Invest. 1993, 1(3): 291-298; Djordjevic B. et al., Acta Oncologica 1998, 37: 735-739; Djordjevic B. et al., Radiat Res 1998, 150: 275-282) allowed the hybrid spheroids to attach to the culture vessel surface allowing a broad range of cancer cells (obscuring the behavior of the cancer stem cells) to spread out and produce colonies. Those early experiments did not use the more stringent 10 division end point. As such, amplifying transit cells of the tumor were dominant in the resulting sensitivity curves. This is clear from the 5-10% plating efficiency using the earlier method vs. the 0.5-0.76% plating efficiency with the nonattachment and the 10 division endpoint of the current patent application, i.e., ˜90% of the colonies from the earlier methods were not cancer stem cells. Thus, there is a need for a method of measuring the proliferative ability of single cancer stem cells.
Although some mouse hematological tumors (leukemias and lymphomas) can be successfully transplanted into syngeneic hosts with one or a few tumor cell(s) [Hewitt H. B. et al., Nature 1959, 183: 1060-1061; Andrews J. R. et al, Radiat Res 1962, 16:76-81; Hill R. P. et al, Brit J Radiol 1973, 46: 167-174], the same is far from true for solid tumors [Djordjevic B. et al., Acta Oncologica 1998, 37: 735-739; Djordjevic B. et al., Radiat Res 1998, 150: 275-282; Djordjevic B. et al., Indian J Exper Biol 2004, 42: 443-447; Al-Hajj M. et al., Proc Natl Acad Sci USA 2003, 100: 3983-3988; Dick J. E., Proc Natl Acad Sci USA 2003, 100: 3547-3549; Kuperwasser C. et al., Cancer Res 2005, 65: 6130-6138; Jordan C. T. et al., Curr Opinion Cell Biol 2004, 16: 708-712.], even those selected by serial transplantation. For single-cell suspensions of solid tumors, the order of 104-106 cells is usually required for the tumor to implant and grow [Elkind M. M. et al., The Radiobiology of Cultured Mammalian Cells, pp. 69-74. Gordon and Breach, New York, 1967]. For in vivo transplantation into syngeneic or immunologically compromised hosts, one could always argue that some minimal immunological response remained which sufficed to explain the large numbers of cells needed to ensure successful tumor implantation and growth. The TD50 (tumor dose to yield 50% of hosts succumbing to the tumor) became the measure of the number of cells needed to produce a tumor, and the ratios of TD50s for treated vs untreated tumor cells became the measure of the tumor cell surviving fractions and survival curves for each treatment modality [Hewitt H. B. et al., Nature 1959, 183: 1060-1061, Djordjevic B. et al., Acta Oncologica 1998, 37: 735-739; Djordjevic B. et al., Radiat Res 1998, 150: 275-282; Hall E. J. et al., Giaccia A J. Radiobiology for the radiologist 6th Edn p 359 Lippincott Williams & Wilkins, Philadelphia, Pa. 200].
An alternative explanation, particularly in the absence of an immunological response, is that only a small fraction of these cells are the cancer stem cells (CSCs) necessary for tumor engraftment and growth. Another factor which may come into play in the low rate of tumor initiation by marker selected CSCs is the need for a stem cell niche (micro-environment) suitable for maintenance of stemness [Schofield, R., Blood Cells 1978, 4: 7-25]. Injection of marker-selected cell populations into a mouse ignores the niche requirement, hoping that some CSCs will find one. Our hybrid spheroids (hs) appear to provide such a niche, enabling a fraction of the tumor cells comparable to that expected to be CSCs, to proliferate extensively (≧ 10 divisions). Presumably injection of such “growth defined hs into immunologically depressed (B and T cell null, NK cell low) xenograft hosts (NOD/SCID mice) may provide the necessary niche (for at least some of the CSCs in the hs) for tumor initiation.
In vivo models are not necessarily better than in vitro ones since “Transplantable tumors in small lab animals tend to be fast growing, undifferentiated, and highly antigenic and are grown as encapsulated tumors in muscle or underneath the skin, not in their sites of origin” [Hall E. J. et al., Giaccia A J. Radiobiology for the radiologist 6th Edn p 359 Lippincott Williams & Wilkins, Philadelphia, Pa. 2006]. Hence, much can be learned from in vitro studies too.
Nevertheless, the value of the confirming in vivo assay for solid tumors is that it tests for the ability of the nascent tumor to grow the necessary vasculature to permit more extensive tumor growth (to 1-2 cm diameter, equivalent to 37-40 divisions by a single CSC in the original hs).
The first attempt to measure the clonogenic survival of tumors in culture as an assay system was that of Salmon and Hamburger [Salmon S. E. et al., Science 1977, 197: 461-463]. Their system of tumor cell growth in soft agar produced sufficient cells to perform an assay in only about 25% of their patient samples and 105 cells had to be plated to get a colony. The low plating efficiency (PE=10−5) made it difficult to claim that the results were representative of the tumor in situ. Typical in vivo PEs of 10−6 in immunologically compromised hosts were no better. An improvement on these systems was based on the Courtney et al. double layer soft-agar method [Djordjevic, B. et al., Cancer Invest 1991, 9(5): 505-512,], used by West et al. [West C. M. L. et al., Br J Cancer 1997, 76: 1184-1190] to measure the sensitivity of cervical cancers to a single dose of 2 Gy. This method obtained results from about three quarters of patients and had a PE of about 0.1% (10−3). A major benefit of the soft-agar methods was supposed to have been that they prevented the growth of stromal fibroblasts, which would otherwise take over the culture. However, Lawton [Lawton P. A. et al., Radiother Oncol 1994, 32(3): 218-225] and Stausbol-Gron et al. [Stausbol-Gron B. et al., Radiother Oncol 1995, 37(2): 87-99; Stausbol-Gron B. et al. Radiother Oncol, 1995, 37(2): 87-99; Stausbol-Gron B. et al. Br J Cancer, 1999, 79(7-8): 1074-84 and Schofield, R., Blood Cells 1978, 4: 7-25] showed that this assumption was incorrect; stromal fibroblasts also proliferate in agar, so that the results were for a mixture of fibroblasts and tumor cells, and not tumor cells only. Moreover, like all single-cell plating assays, there is a fundamental problem that soft-agar assays lack physiological relevance to in situ tumors, because they lack the three dimensional (3D) cell-cell contact seen in tumors, and the lack of a stem cell niche may be critical [Schofield, R., Blood Cells 1978, 4: 7-25; Stausbol-Gron B. et al., Radiother Oncol 1995, 37(2): 87-99; Stausbol-Gron B. et al. Radiother Oncol, 1995, 37(2): 87-99; Stausbol-Gron B. et al. Br J Cancer, 1999, 79(7-8): 1074-84]. This may be why no one has yet correctly predicted individual patient outcomes using these assays [West C. M. L. et al., Br J Cancer 1997, 76: 1184-1190; Hill R. P. Cancer Res 2006, 66: 1891-1895; Kern S. E. et al., Cancer Res 2007, 67; 8985-8988].
To alleviate these problems, an in vivo-like system, the hs assay has been developed [Djordjevic B. et al., Acta Oncologica 1998, 37: 735-739; Djordjevic B. et al., Acta Oncologica, 2006, 45, 412-420; Djordjevic B. et al., Radiat. Res. 53rd Annual Meeting Program Book p 99 (PS179), Nov. 6-9, 2006, Philadelphia, Pa.; Hill R. P. et al., Cancer Res 2006, 66: 1891-1895; Kern S. E. et al., Cancer Res 2007, 67: 8985-8988;
Djordjevic B. et al., Radiat Environ Biophys 1990, 29: 31-46; Lange C. S. et al., Int J Radiat Oncol Biol Phys 1992, 24(3): 511-518; Djordjevic B. et al., Cancer Invest 1991, 9(5): 505-512; Djordjevic B. et al., Cancer Invest. 1993, 11(3): 291-298], which is suitable for testing primary tumor cells (see below). This system exhibits a much higher PE, ˜0.5-2% [Djordjevic B. et al., Acta Oncologica, 2006, 45: 412-420], with almost all samples producing sufficient colonies for assay [Djordjevic B. et al., Acta Oncologica, 2006, 45: 412-420; Djordjevic B. et al., Radiat. Res. 53rd Annual Meeting Program Book p 99 (PS179), Nov. 6-9, 2006, Philadelphia, Pa.; Hill R. P. et al., Cancer Res 2006, 66: 1891-1895]. This PE is consistent with the hypothesis that what formed colonies in vitro were the cancer stem cells (CSCs). Furthermore, in the hybrid spheroid system, cells are enveloped in a three-dimensional (3D) agglomerate of cells, initially providing an analog of the stem cell niche, and after treatment exhibiting all the mutual influences on survival. This is important, as it is becoming clear that the initial growth of CSCs depends on having an appropriate niche (plating of 40,000 single tumor cells fails to yield even one colony when ˜400 would have been expected from the hs PE), and that the survival of tumor cells and the functionality of various tissues surrounding tumors are determined not only by the direct impact of inactivating agents, but also by the now well recognized Bystander Effect (BE) (see Choo A. et al., J Biotechnol 2006, 122: 130-141; Azzam E. I. et al., Hum Exp Toxicol 2004, 23: 61-65; Iyer R. et al., Arch Biochem Biophys 2000, 376: 14-25; Seymour C. B. et al., Radiat Res 2000, 153: 508-511; Ishii K. et al., Int J Radiat Biol 1996, 69: 291-299; Bishayee A. et al., Radiat Res 1999, 152: 88-97; Azzam E. I. et al., Radiat Res 1998, 150: 497-504; Djordjevic B., BioEssays 2000, 22: 286-290).